Method for Promoting Respiratory Burst Activity Promoter and Immunostimulating in Type 2 Diabetic Patients

ABSTRACT

To confirm the safety of a fermented  papaya  preparation in type 2 diabetic patients while determining its in vivo effect on respiratory burst in human peripheral-blood mononuclear cells to find further health advantages for the type 2 diabetic patients. A respiratory burst activity promoter for improving the production of reactive oxygen species (ROS) induced by NADPH oxidase and promoting respiratory burst activity in type 2 diabetic patients, comprising a fermented  papaya  preparation as an active ingredient.

TECHNICAL FIELD

The present invention relates to a respiratory burst activity promoterfor promoting respiratory burst activity, and an immunostimulant.Specifically, the invention relates to a respiratory burst activitypromoter for improving the production of reactive oxygen species (ROS)induced by NADPH oxidase and promoting respiratory burst activity intype 2 diabetic patients, comprising a fermented papaya preparation(FPP) as an active ingredient, and an immunostimulant, based onimproving the production of reactive oxygen species (ROS) induced byNADPH oxidase and promoting respiratory burst activity in type 2diabetic patients, comprising FPP as an active ingredient.

BACKGROUND ART

FPP produced by fermenting the unripe fruit of Carica papaya Linntogether with sugar using edible yeast contains increased maltose andmaltotriose in a mixture with saliva compared to that in a mixture withwater.

The oral ingestion of FPP is expected to serve to improve the intestinalenvironment by increased oligosaccharides and also expected to suppressan increase in the blood glucose level and promote wound healing in type2 diabetic patients (Patent Literature 1). FPP is also known to have anantioxidant property and be effective on symptoms associated with aging(Non Patent Literature 1).

A chronic wound is an important problem in health for diabetic patients(Non Patent Literature 2). The present inventors recently reported, forthe first time, evidence that FPP had the possibility of particularlyinfluencing macrophage response in the wound area and subsequentangiogenic response to improve the surface wound of diabetic patients(Non Patent Literature 3). The present inventors also showed that FPPpromoted respiratory burst activity ex vivo in human peripheral bloodmononuclear cells (PBMC) of patients with type 2 diabetes (T2D),resulting in NADPH oxidase-dependent improvement (Non Patent Literature4).

ATP synthesis and mitochondrial respiration are two pathways forming thecore of cellular metabolism. Respiration consists of the oxidation ofmitochondrial NADPH by oxygen. The formation of NADPH by oxygen works inconjunction with the electron transport chain producing anelectrochemical gradient of protons consisting of membrane potential andpH gradient (Non Patent Literature 5). The human peripheral bloodmononuclear cells (PBMC) of patients with type 2 diabetes (T2D) resultin a reduction in the respiratory burst activity, which increases therisk of infectious disease.

In recent years, the importance of two ROSs, i.e., “good ROS vs bad ROS”has also been in the spotlight (Non Patent Literature 6).

CITATION LIST Patent Literature

-   Patent Literature 1: Japanese Patent Application Laid-Open No.    2011-041478

Non Patent Literature

-   Non Patent Literature 1: Aruoma O I, Hayashi Y, Marotta F, Mantello    P, Rachmilewitz E, Montagnier L. Applications and bioefficacy of the    functional food supplement fermented papaya preparation. Toxicology    278: 6-16, 2010.-   Non Patent Literature 2: Sen C K, Gordillo G M, Roy S, Kirsner R,    Lambert L, Hunt T K, Gottrup F, Gurtner G C, Longaker M T. Human    Skin Wounds: A Major and Snowballing Threat to Public Health and the    Economy. Wound Repair Regen in press, 2009.-   Non Patent Literature 3: Collard E, Roy S. Improved function of    diabetic wound-site macrophages and accelerated wound closure in    response to oral supplementation of a fermented papaya preparation.    Antioxid Redox Signal 13: 599-606, 2010.-   Non Patent Literature 4: Dickerson R, Deshpande B, Gnyawali U, Lynch    D, Gordillo G M, Schuster D, Osei K, Roy S. Correction of aberrant    NADPH oxidase activity in blood-derived mononuclear cells from type    II diabetes mellitus patients by a naturally fermented papaya    preparation. Antioxid Redox Signal 17: 485-91, 2012.-   Non Patent Literature 5: Brown G C. Control of respiration and ATP    synthesis in mammalian mitochondria and cells. Biochem J 284 (Pt 1):    1-13, 1992.-   Non Patent Literature 6: Ray L B. Good ROS Versus Bad ROS. Sci.    Signal. 3: ec218, 2010.

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to confirm the safety of FPP intype 2 diabetic patients while determining its in vivo effect onrespiratory burst in human peripheral-blood mononuclear cells to findfurther health advantages for the type 2 diabetic patients.

Solution to Problem

As a result of various studies for achieving the above object, thepresent inventors have found that FPP improves the production ofreactive oxygen species (ROS) induced by NADPH oxidase and promotesrespiratory burst activity in type 2 diabetic patients.

Thus, the present invention provides a respiratory burst activitypromoter for improving the production of reactive oxygen species (ROS)induced by NADPH oxidase and promoting respiratory burst activity intype 2 diabetic patients, comprising FPP as an active ingredient. Theinvention also provides an immunostimulant, based on improving theproduction of reactive oxygen species (ROS) induced by NADPH oxidase andpromoting respiratory burst activity in type 2 diabetic patients,comprising FPP as an active ingredient.

Advantageous Effects of Invention

According to the respiratory burst activity promoter of the presentinvention, respiratory burst activity can be promoted without adverselyaffecting the blood glucose level of patients with type 2 diabetes(T2D). This is useful for T2D patients since particularly in T2Dpatients in whom the respiratory burst activity is reduced, “good ROS”(respiratory burst) is promoted and “bad ROS” (oxidative stress) isremoved. This enables an increase in the immunity of type 2 diabeticpatients.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a series of graphs showing the results of confirming thesafety of FPP in T2D patients. The oral ingestion of FPP little affectedfasting blood glucose (A and D), HbA1c (B and E), or total cholesterol(C and F) in T2D patients. Data are presented as a mean±standarddeviation (n=15).

FIG. 2 is a series of graphs showing that the oral ingestion of FPP hadthe effect of improving respiratory burst activity in peripheral-bloodmononuclear cells of T2D patients. FPP improved the reduced respiratoryburst activity induced by T2D but did not affect systemic oxidativestress. “A” indicates a fold change in superoxide anion production overbase line, and data are presented as a mean±standard deviation (n=14). *indicates p<0.05. “B” indicates a change (%) in the protein carbonyllevel over base line, and data are presented as a mean±standarddeviation (n=10). “C” indicates a change (%) in the 4-hydroxynonenal(HNE-4) level over base line, and data are presented as a mean±standarddeviation (n=17).

FIG. 3 is a series of graphs showing the effect of promotingintracellular ATP and NADPH production by FPP. Data are presented as amean±standard deviation (n=4). * indicates p<0.05 against NG.

FIG. 4 is a series of graphs showing the effect of promotingmitochondrial membrane potential and oxygen consumption by FPP. “A”indicates the results of flow cytometry. “B” is a graph showing theratio of depolarized cells to polarized cells (FL2-h/FL1-h); § indicatesp<0.05 against glucose-depleted cells; and * indicates p<0.05 against NG“C” is a schematic of tracing the measurement of state IV and uncoupledrespiration in NG and NG+FPP. “D” is a graph showing the ratio ofuncoupled rate to state IV rate. * indicates p<0.05 against NG (n=3).

DESCRIPTION OF EMBODIMENTS

The respiratory burst activity promoter of the present inventioncomprises FPP as an active ingredient, and improves the production ofreactive oxygen species (ROS) induced by NADPH oxidase and promotesrespiratory burst activity in type 2 diabetic patients. Theimmunostimulant of the present invention comprises FPP as an activeingredient, and is based on improving the production of reactive oxygenspecies (ROS) induced by NADPH oxidase and promoting respiratory burstactivity in type 2 diabetic patients.

As described above, FPP is a papaya-derived fermented product producedby fermenting the unripe fruit of Carica papaya Linn together with sugarusing edible yeast.

It is preferable for FPP to be one produced by Osato Laboratory Inc. andsold by Osato International Inc. (Patent Literature 1 and Non PatentLiterature 1). The FPP can be obtained as “FPP Fermented PapayaPreparation” (R) or “Immun' Age” (R). The FPP is produced in a factoryreceiving ISO 9001: 2008, ISO 14001: 2004, and ISO 22000: 2005certifications and gaining the certification of FSSC 22000 as a mostrigorous food safety standard in Europe and the United States as a firstreceiver in JIA, Japan, and is guaranteed in terms of quality,environment aspect, and safety.

The method for producing FPP is described, for example, in PatentLiterature 1. According to analysis by Japan Food Research Laboratories,91.2 g in 100 g of FPP is carbohydrate, and FPP also contains smallamounts of protein (0.3 g), lipid (0.2 g), potassium (14.9 mg), andwater (8.5 g) (lot. no. 091; analysis certificate dated May 27, 2014).

FPP may be properly prepared in various forms, such as granules,powders, and subtle granules in a manner suitable for oral ingestion,and, in preparation, additives, for example, an excipient, a binder, alubricant, may be properly added.

The usage amount of FPP as an active ingredient may be 0.5 to 30 g/dayper adult having a body weight of 70 kg, preferably 1 to 20 g/day, morepreferably 3 to 15 g/day, most preferably 3 to 9 g/day.

The respiratory burst activity promoter of the present invention canpromote respiratory burst activity without adversely affecting the bloodglucose level of type 2 diabetic (T2D) patients in whom the respiratoryburst activity is reduced. From this, health effects, such asimmunopotentiation, can be expected in T2D patients. FPP is the world'sfirst substance capable of enhancing immunity in diabetic patientswithout changing the blood glucose level despite being a carbohydrate.The basis therefor is that the substance increases respiratory burstactivity by improving ROS production due to NADPH oxidase induction indiabetic patients.

EXAMPLES Materials and Methods

(1) Human Subject and Sample Collection

The clinical trial in humans was approved by the institutional reviewboard of Ohio State University (OSU). The adult subjects in the clinicaltrial were those who were clinically diagnosed as type 2 diabetesmellitus, had well-controlled glucose levels, and had an HbA1c value of7% or less. The subjects were recruited in a diabetes clinic located inthe Ohio State University Wexner Medical Center, and patients having asuppressed immune response or taking a peroxisomalproliferator-activated receptor-γ agent were excluded from the trial.Peripheral blood (60 cc) was collected by venipuncture, and 10 cc of theblood was sent to the Ohio State University Wexner Medical CenterClinical Laboratories and subjected to blood chemical examination. Theremaining blood was used for the separation of peripheral-bloodmononuclear cells (PBMC) and plasma. PBMC was promptly used forrespiratory burst assay. The plasma from the patients was separated fromthe whole blood, centrifuged, snap-frozen in liquid nitrogen, and storedat −80° C. until all samples were collected, and the samples weresubsequently analyzed together.

(2) Separation and Culture of Human Peripheral-Blood Mononuclear Cell

Fresh blood was 1:1 diluted with Dulbecco's phosphate buffered saline(DPBS; Gibco/Life Technologies, Carlsbad, Calif.). Humanperipheral-blood mononuclear cells (PBMC) were separated by Ficolldensity centrifugation and subsequent sorting with magnetic beads coatedwith anti-CD14 (Miltenyi Biotec, Auburn, Calif., USA).

(3) Measurement of Superoxide Anion

The promoted production of superoxide anion (O₂ ⁻) was detected using achemiluminescence LumiMax (R) superoxide anion measurement kit(Stratagene, La Jolla, Calif.) according to a maker-recommendedprotocol. The production of reactive oxygen species (ROS) in respiratoryburst in PBMC was measured after stimulation with 1 μg/ml of phorbolmyristate acetate (PMA). A human promyelocytic leukemia cell line(HL-60) was used as a control.

(4) Cell Culture

A human monocytic cell line, THP-1 cells, was cultured for 48 hours inRPMI1640 glucose-free medium (Life Technologies, Carlsbad, Calif.)containing 10% fetal bovine serum (FBS) and 1% antibiotic/antifungalagent (AA) to remove glucose from the cells. Then, to recovercarbohydrate in the cells, the above glucose-free medium wassupplemented by the following substrate: a normal glucose level (NG) (11mM or 2 mg/mL glucose in the medium), a high glucose level (HG) (20 mMor 3.9 mg/mL glucose in the medium), FPP (2.9 mg/mL FPP in the medium),or NG+FPP (11 mM or 2 mg/mL glucose and 2.9 mg/mL FPP in the medium).The supplemented medium was subjected to sterile filtration with a0.22-μm vacuum filter system (Millipore, Billerica, Mass., USA), andsubsequently added to the THP-1 cells from which glucose was removed.The cells for the substance groups were cultured together with therespective substrates for 24 hours and subjected to assay.

HL-60 cells were cultured in high-glucose DMEM which contained sodiumpyruvate (Life Technologies, Carlsbad, Calif., USA) and to which 20% FBSand 1% AA were added.

(5) Measurement of Metabolite and Oxidative Stress

The concentration of NADP/NADPH was measured using a NADP/NADPHquantification kit (Sigma-Aldrich, St. Louis, Mo., USA) according to amaker-recommended protocol. THP-1 cells were washed with cold DPBS afterrecovering carbohydrate for 24 hours. The cells were lysed by repeating2 cycles of freezing and thawing. To remove an NADPH-consuming enzyme,the lysate was filtered using a 10-kDa ultrafiltration spin column(Amicon/Millipore, Billerica, Mass., USA). To detect NADPH, a samplecontaining total NADP was heated at 60° C. for 30 minutes and NADP wasdigested to leave only NADPH. The total NADP sample and the NADPH samplewere incubated together with an NADP cycling enzyme at room temperaturefor 5 minutes to convert NADP to NADPH. A developer for colorimetricanalysis was added to the sample, which was then incubated at roomtemperature for 1 hour. Absorption at 450 nm was measured using anautomated microplate (Model Synergy 2, BioTek, Winooski, Vt., USA).

The ATP concentration was measured using an ATP assay kit (Abeam,Cambridge, Mass., USA) according to a maker-recommended protocol. THP-1cells were washed with cold DPBS after recovering carbohydrate for 24hours. Protein was removed from the sample using ice-cold 4 M perchloricacid (PCA). An equal amount of 2 M potassium hydroxide (KOH) was addedto neutralize the sample to pH 6.8-7.2 to precipitate excess PCA. Thesample was incubated for 30 minutes and subjected to colorimetric assay,and then absorption at 570 nm was measured.

Protein carbonyl was measured using Protein Carbonyl Content Assay Kit(Abeam, Cambridge, Mass., USA) according to a maker-recommendedprotocol.

4-Hydroxynonenal (HNE-4) was measured using OxiSelect™ ENE AdductCompetitive ELISA kit (Cell Biolabs, Inc. San Diego, Calif., USA)according to a maker-recommended protocol.

(6) Mitochondrial Membrane Potential Measurement

THP-1 cells were cultured together with cationic JC-1 dye (LifeTechnologies, Carlsbad, Calif.) capable of accumulating depending on thepotential difference in mitochondria, for 20 minutes under conventionalconditions. The polarized state of the cells cultured with the abovesubstrate group was measured by measuring an emission shift from green(about 525 nm, FL1-H channel) to red (about 590 nm, FL2-H channel). Thereduced emission ratio of red to green indicates depolarization or lowmembrane potential. The emission ratio of red to green entirely dependson the membrane potential and is not affected by the size, density, orshape of mitochondria. Measurements were collected using flow cytometry(model C6 Flow Cytometer, Accuri, Ann Arbor, Mich., USA) after 25 Kevents per sample.

(7) Oxygraph Measurement of Oxygen Consumption

To measure the consumption of oxygen (O₂), stage IV and uncoupledrespirations were measured. Cells were subjected to permealizationtreatment using digitonin (4 μg/10⁶ cells). The respiratory mediumcontains 230 mM mannitol, 70 mM sucrose, 3 mM HEPES (pH 7.4), and 10 mMsuccinic acid ester. The succinic acid ester was first oxidized withcomplex II. Then, oligomycin (2 mM) was added to inhibit complex V (ATPsynthase) to prevent the formation of ATP from endogenous ADP (state IVrespiration). The maximal respiration was generated by adding carbonylcyanide m-chlorophenylhydrazone (CCP, 1.5 μM) as an uncoupling agent tochemically extinguish the membrane potential. Finally, antimycin A (1.5mM) was added to suppress complex III. This limits the transfer ofelectrons to cytochrome c and the subsequent reduction of O₂ to H₂O bycomplex IV, resulting in the suppression of oxygen consumption. Theoxygen consumption rate was measured using a Clark's oxygen electrodeand an oxygen monitor (Yellow Springs Instrument, Yellow Springs, Ohio,USA). Oxygen consumption was calculated by dividing the uncoupled rateby the state IV rate.

(8) Statistics

In vitro data are reported as the mean±standard error of triplicate topentaplicate experiments. The comparison between a plurality of thegroups was tested by variance analysis (ANOVA). p<0.05 indicatesstatistically significant difference. Data from 17 adult subjects (n=17)were presented for the human T2D trial (Table 1).

Example 1 Safety of FPP in T2D Patient

Before examining the in vivo effect of FPP in T2D patients, the safetyof FPP in T2D patients was first confirmed.

In 17 patients diagnosed as T2D, blood before the start of FPP ingestion(base line (BL)) was collected in OSU Diabetic Clinic, and FPP (9 g/day)was ingested for subsequent 6 weeks, during which they came 2 times tothe clinic. The test was completed by 2 times visiting to the clinicduring subsequent wash-out. With every visiting to the clinic,respiratory burst ROS production, fasting blood glucose, lipid value,glycated hemoglobin (HbA1c), and lipid/protein peroxidation reactionwere measured. Peripheral blood was collected from T2D patients in OSUDiabetic Clinic (see Table 1). For T2D patients, the period of 6-weekFPP (9 g/day) ingestion and 2-week wash-out was provided.

TABLE 1 Patient Statistics Total Number of Recruitments (n) 22 Number ofCompleted Study (n) 17 Age (Years) 56.4 ± 12  Gender-Males 12Gender-Females 10 Ethnicity-White 6 Ethnicity-Black 12 Ethnicity-Asian 4HbA1c (%)   7 ± 0.8 BMI (kg/m²) 32.8 ± 4.9

The return of an empty used bag for FPP was defined as patient'scompliance, and the average compliance rate was 90%. Fasting bloodglucose, glycated hemoglobin (HbA1c), and cholesterol levels weremeasured during base-line, ingestion, and wash-out.

The results are shown in FIG. 1. FIG. 1 suggested that since theingestion of FPP did not affect blood glucose and total lipid levels,FPP could be safely ingested by T2D patients. As shown by the HbA1clevel, no long-term effect on the blood glucose level was observed to bechanged throughout this study period.

The oral ingestion of FPP remarkably induced respiratory burst ROSproduction in PBMC, while not affecting the blood components duringingestion. FPP has a long track record on the safety of human ingestion.This study has originality in terms of having, for the first time,investigated the influence of FPP on blood glucose, HbA1c, and totallipid levels in T2D patients. These results showed that FPP was high intolerability for T2D patients despite its sweetness and its carbohydratecomposition.

Example 2 Improvement of Respiratory Burst Activity in Peripheral-BloodMononuclear Cell of T2D Patient by FPP

The present inventors previously reported that the ex vivo treatment ofhuman peripheral-blood mononuclear cells (PBMC) with FPP improved areduction in respiratory burst due to T2D when stimulated with phorbol12-myristate 13-acetate (PMA) (Non Patent Literature 4).

To verify whether or not the in vivo oral ingestion of FPP could improverespiratory burst activity in monocytes, peripheral-blood mononuclearcells (PBMC) were collected from T2D patients. PBMC were collected fromT2D subjects during base-line, 2 and 6 weeks after starting theingestion of FPP, and 2 weeks after wash-out. After stimulation with PMA(1 μg/ml) for 30 minutes, the production of reactive oxygen species(ROS) superoxide anion during respiratory burst in PBMC was measured bydetection by chemiluminescence. Protein carbonyl and 4-hydroxynonenal(HNE-4) were measured by ELISA, as described above. The fresh plasma wasone which was snap-frozen in liquid nitrogen, and stored until allsamples were collected. All samples were simultaneously measured.

The results are shown in FIG. 2. It was revealed from FIG. 2 that theproduction of ROS was significantly promoted compared to that duringbase-line in the patients (A in FIG. 2). It was previously reported thatthe ingestion of α-tocopherol as an antioxidant decreased the release ofsuperoxide anion in monocytes (Devaraj S, Tang R, Adams-Huet B, HarrisA, Seenivasan T, de Lemos J A, Jialal I. Effect of high-dosealpha-tocopherol supplementation on biomarkers of oxidative stress andinflammation and carotid atherosclerosis in patients with coronaryartery disease. Am J Clin Nutr 86: 1392-8, 2007.).

To verify whether or not the production of ROS through NADPH oxidase dueto FPP increased systemic oxidative stress, the influence of FPP oncarbonyl protein and 4-hydroxynonenal (HNE-4) as a lipid peroxidereaction marker was measured with every visiting to the clinic. FPPincreased necessary respiratory burst ROS in T2D patients but did notadversely affect oxidative stress throughout the FPP ingestion period (Bto C in FIG. 2).

The oral ingestion of 6 g/day of FPP for 14 weeks was reported tosignificantly reduce the hemolysis rate and decrease the accumulation ofprotein carbonyl in the serum of patients with prediabetes (Somanah J,Bourdon E, Rondeau P, Bahorun T, Aruoma O I. Relationship betweenfermented papaya preparation supplementation, erythrocyte integrity andantioxidant status in pre-diabetics. Food Chem Toxicol 65: 12-7, 2014.).

Based on these observations, the present inventors presume that theingestion of FPP can improve innate immune response in diabeticpatients.

Example 3 Promotion of Intracellular Production of ATP and NADPH by FPP

The hyperphosphorylation of oxidase subunit p47 (PHOX) occurs when NADPHoxidase is active in intact cells. The present inventors previouslyreported that p47 (PHOX) was upregulated in PBMC ex vivo given FPP.

As described in [Materials and Methods], the human monocytic cell line,THP-1 cells, was cultured in the glucose-free medium for 48 hours, and,after removing glucose from the cells, cultured for 24 hours in a mediumcontaining a normal glucose level (NG), a high glucose level (HG), FPP,or NG+FPP. Thereafter, the cells for the substance groups were measuredfor intracellular ATP, NADP, and NADPH by ELISA.

The results are shown in FIG. 3. ATP is necessary for phosphorylationreaction. In the human monocytic cell line, THP-1 cells, the ATP levelwas 75% higher for the glucose medium containing FPP (NG+FPP) than thatfor only the glucose medium (NG), suggesting that FPP promoted ATPproduction in the monocytes (A in FIG. 3). These results are unexpectedand new in that the sweet dietary supplement can increase intracellularATP production. Since the same results were not observed for the highglucose medium (HG), it was suggested that the increase in the ATP leveldid not depend on glucose contained in FPP and a particular component inFPP promoted the intracellular production of ATP in monocytes.

NADPH oxidase in leukocytes consumes NADPH to catalyze superoxide anionproduction. Interestingly, the treatment of THP-1 cells with FPP onlyslightly increased total NADP but increased the NADPH level of the cellsby 30% (B to C in FIG. 3).

A dramatic transition was observed in the percentage of NADPH inNADPH/NADP, and it was suggested the possibility that the increasedproduction of respiratory burst ROS through NADPH oxidase in T2D is aconsequence of the increased level of ATP and NADPH in the cells (D inFIG. 3).

These results suggested that FPP promoted NADPH oxidase activity byincreasing cellular ATP and NADPH levels.

Example 4 Increase in Mitochondrial Membrane Potential and RespiratoryActivation by FPP

ATP synthesis and mitochondrial respiration are two pathways forming thecore of cellular metabolism. Respiration consists of the oxidation ofmitochondrial NADPH by oxygen. The formation of NADPH by oxygen works inconjunction with the electron transport chain producing anelectrochemical gradient of protons consisting of membrane potential andpH gradient.

ATP synthesis in mitochondria works in conjunction with ATP synthesis inthe matrix of mitochondria by the penetration of protons into themembrane. ATP (adenosine triphosphate) is the major energy source forcells, and used for processes in most principal cells, includingrespiratory burst by NADPH oxidase.

To verify whether the ATP level of cells increases due to mitochondrialmembrane potential (MMP) and respiration, the mitochondrial membranepotential was measured. As in Example 3, the human monocytic cell line,THP-1 cells, was cultured in the glucose-free medium for 48 hours, and,after removing glucose from the cells, cultured for 24 hours in a mediumcontaining a normal glucose level (NG), a high glucose level (HG), FPP,or NG+FPP. Thereafter, the cells for the substance groups were measuredfor mitochondrial membrane potential and oxygen consumption as describedin [Materials and Methods]. The mitochondrial membrane potential wasmeasured by JC-1 dye and flow cytometry.

The results are shown in FIG. 4. An increase in red fluorescenceemission was observed in the group of monocytic cells cultured underconditions of FPP and the normal glucose level (NG+FPP) (A in FIG. 4).An about 20% increase in MMP was observed in monocytic cells culturedunder conditions of NG+FPP compared to that in monocytic cells culturedunder conditions of the normal glucose level containing no FPP (NG) (Bin FIG. 4).

To support the hypothesis of the present inventors that theadministration of FPP increases mitochondrial respiration and therebyincreases ATP production, the oxygen consumption of monocytic cells inthe presence and absence of FPP administration was measured using aClark's oxygen electrode monitor (C in FIG. 4).

The ratio of the uncoupled respiration rate to the state IV respirationrate for NG+FPP increased compared to that for NG, showing that theoxygen consumption of the cultured cells increased (D in FIG. 4).

Taken together, FPP may increase the supply of ATP and NADPH byactivating mitochondrial respiration, resulting in the promotion ofrespiratory burst in PBMC of T2D patients. FPP has a long history,during which it has been safely consumed by humans; with reference tothe clinical data from this study, FPP is expected to be effective oninnate immune responses in T2D patients.

In diabetes, the blood glucose level increases since abnormal glucosemetabolism fails to convert glucose in the body to energy, and thusperipheral circulation is made poor and immunity is depressed. Thisclinical study was the world's first clinical study directed, for thefirst time, toward an improvement in immunity and an enhancement inenergy metabolism using FPP despite that FPP was made of carbohydrate.In addition, the present inventors present the new results that FPPstimulates mitochondrial respiration and promotes cellular ATPproduction without adversely affecting the blood glucose level of T2Dpatients.

A previous paper has reported that FPP has the possibility of havingantioxidant activity. In recent years, the importance of two ROSs, i.e.,“good ROS vs bad ROS” has also been in the spotlight (Non PatentLiterature 6). FPP is given to significantly improve respiratory burstactivity in monocytes. The possibility is also presented for the firsttime that FPP may be useful for T2D patients by serving two purposes inthe physiological function of reactive oxygen species (ROS), i.e.,promoting “good ROS” (respiratory burst) and removing “bad ROS”(oxidative stress).

1-2. (canceled)
 3. A method of promoting respiratory burst activity in a type 2 diabetic patient, comprising a step of orally administering to the type 2 diabetic patient a fermented papaya preparation in an amount effective to improve production of reactive oxygen species (ROS) induced by NADPH oxidase.
 4. A method of immunostimulating in a type 2 diabetic patient, comprising a step of orally administering to the type 2 diabetic patient a fermented papaya preparation in an amount effective to improve production of reactive oxygen species (ROS) induced by NADPH oxidase and promote respiratory burst activity. 